Iodinated estradiols, intermediate products and methods of production

ABSTRACT

A method of preparation of estradiol derivatives labeled by iodine, especially  123  I, substitution at C-16 are synthesized according to the present invention. A triflate intermediate is prepared from which either 16-alpha- 123  I-17-beta-estradiol or 16-beta- 123  I-17-beta-estradiol are prepared by described methods. Both 16-alpha- 123  I- and 16-beta- 123  I-17-beta-estradiol made according to the methods described herein have a high relative specific acitvity. The methods are sufficiently rapid so that the relatively short half-life of  123  I is readily accommodated without substantial radioactive decay of the label.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 663,571,filed Oct. 22, 1984, now abandoned.

BACKGROUND OF THE INVENTION

Certain research related to the subject matter of the presentapplication was conducted under monetary grants from the United Statesof America and a paid up, nonexclusive, irrevocable and non-transferablelicense is hereby granted to the United States of America forgovernmental purposes.

This invention relates to estradiol derivatives, their syntheses and thepreparation of their synthetic intermediates or precursors. Inparticular, this invention relates to the preparation of certainsubstituted estradiols which have a specific, preferred sterochemistry.

The term "estradiol", as used herein, refers to compounds having thefollowing general structural formula: ##STR1##

Estradiols similar to the compound I are, generically,estra-1,3,5-(10)-triene-3,17-diols. In the above drawing the carbons inthe estradiol structure are numbered according to the generallyrecognized nomenclature system for steroids, with the hydroxysubstituents located at the 3- and 17-positions. It will be understoodthat the substituent at the 17 position may have either of twoorientations, referred to as alpha and beta. Generally, an alphasubstituent is one which projects beneath the plane of the drawing shownabove and a beta substituent is one which projects above the plane ofthe steroidal ring drawing. The same would be true for substituentslocated at C-16.

Substance II shown below is a form of estradiol generally medicallyrecognized to be of particular importance and has a17-hydroxy-substituent located beta. A commonly used name for thissubstance is estra-1,3,5(10)-triene-3,17-beta-diol (17-beta-estradiol orbetaestradiol). ##STR2##

In general, many estradiols are naturally occuring substances whosederivatives have been found to have medicinal use. For example, the3-methyl ether has been used for replacement therapy in estrogendeficiency. Also, certain radioactively labeled estradiols may be usedin estrogen receptor assays. In particular, the tritiated,iodine-125(I-125) and bromine-77(Br-77) labeled substances have beentested.

It is known that estrogen receptors, i.e. binding substrates forestradiols, may be found in certain animal tissues. It has also beenfound that the presence of estrogen receptors may be connected withcertain abnormalities in the tissue. Estradiols, if properly labeled,may be utilized to detect the presence of these estrogen receptors intissue. The medical profession generally theorizes that these estrogenreceptor analyses may be conducted in vitro or in vivo. Further,applicants foresee that appropriately labeled estradiols may be utilizedto deliver a radioactive iodine, especially ¹²³ I, to a site in tissue,in order to promote a therapeutic effect.

In particular, certain 17-beta-estradiols, which have been substitutedat the 16-position, are generally thought to have an affinity forestrogen receptors which is both significant and useful in performingestrogen receptor analyses, including assays and imaging. It is foreseenthat numerous 16-substituted-17-beta-estradiols may be of importance,particularly those in which the 16-substituent is a halogen and mostimportantly when the halogen is radioactive iodine, especially ¹²³ I,which has a relatively quick half life and high energy radiationassociated with decay thereof. It is foreseen that both16-alpha-16-beta-substituted-estradiols may be of use. However, for anygiven substituent, the affinity of the16-alpha-substituted-17-beta-estradiol, for estrogen receptors, islikely to differ from that for the analogous16-beta-substituted-17-beta-estradiol.

Since both the 16-alpha-substituted and16-beta-substituted-17-beta-estradiols are foreseen to have utility, itis prefered that methods of syntheses of each be developed. It ispreferable that each synthetic scheme yield a desired isomersubstantially stereospecifically, so that problems of purification andproblems from the differences of affinity of the two isomers for bindingsites, are avoided. Thus, two general synthetic schemes are needed, onewhich provides 16-alpha-substituted compound with very little16-beta-substituted compound being present, and a second generalreaction methodology which yields 16-beta-substituted compound with verylittle 16-alpha-substituted compound being present.

It is readily seen that it would be most desirable to develop a singlesynthetic precursor or intermediate from which either the 16-alpha- orthe 16-beta-substituted compound can be relatively very rapidly andeasily formed. That is, given a supply of the synthetic intermediate asynthesis laboratory could easily prepare whichever 16substitutedcompound is desired. It is particularly desirable to have alternatesynthetic schemes which are relatively easy to conduct and which areboth very rapid and very efficient and which produce a relatively highpercentage of the desired final product.

With present technology, two basic methods of detection of labeledestradiols are most available. In one, a radioactive substituent isintroduced into the molecule and standard methods of radioisotopedetection are utilized to determine the presence of the labeledestradiol in the animal tissue, either in vitro or in vivo. In theother, nuclear magnetic resonance (NMR) methods are utilized to detectcertain nucleii, and generally non-radioactive labels may be used. Atthe present time, only radioisotope techniques are widely available butit is foreseen that other methods may be come more available in thefuture.

If a radioactive isotope is used as the label, then thestereospecificity of the reactions leading to the synthesis of the16-substituted-17-beta-estradiol may be critical. Also, efficiency ofthe reaction, in terms of product yield and the length of time it takesto introduce the radioactive isotope into the molecule and then isolatethe desired product for diagnostic or therapeutic use, may be veryimportant.

The importance of the stereospecificity is easily understood.Radioactive labels are very expensive and if the reaction is notsufficiently stereospecific large amounts of the label may be lost inundesired products. Also, if the undesired products are to be discardedthere may be problems with dangerous residual waste-productradioactivity. Finally, side products might not be easily separable fromthe desired isomer, and they can interfere with the certainty of assayand imaging data collected when the product is used in medicinalanalyses.

If the product yields are not sufficiently high, much of the radioactiveisotope may not be incorporated into a useful product, again wastingexpensive isotope.

If the reactions involved in the introduction of the radioisotope intothe molecule, when coupled with any further reactions or purificationsnecessary to isolate the desired radioactive products, are notsufficiently rapid, special problems may be encountered. It will beunderstood that the radioactive label is constantly decaying; and, ifthe isotope has a sufficiently short half-life, it must be introducedinto the molecule rapidly, and the compound must be isolated forbiological use relatively rapidly, or the isotope will have passedthrough sufficient half-lives to produce so little "hot" or radioactivesubstrate that detection may be difficult.

In some instances the radioisotope used may be contaminated with smallamounts of other radioisotopes of the same compound. For example,Iodine-123 (I-123) as it is currently made, is often contaminated withsome Iodine-124 (I-124). The half-life for I-123 is about 13.3 hourswhereas the half-life for I-124 is about 4.2 days. I-123 is readilydetectable and gives clear images whereas I-124 generally causes somescattering and images of low resolution. Consider what happens if amixture of 99 to 1 I-123 to I-124, is utilized to label a substrate. Ifthe reaction takes too long, for example 26 hours, for introduction ofthe isotope into the substrate molecule and isolation of the desiredproduct, then the I-123 will pass through about two half-lives and onlyabout 25% of it will be left. The I-124, however, will have barely begundecaying and nearly 100% of it will be left. The ratio of I-123 toI-124, after the 26 hours, will have changed to approximately 24 to 1.It is readily seen that this enhancement of the amount of I-124 present,by ratio, may cause difficulty since the I-124 might make resolution ofimages difficult. Also, if much of the isotope mixture must be given toaccommodate imaging of I-123, residual radioactivity from the I-124component, with its long halflife, may be a problem. These types ofproblem are usually present whenever an isotope of short half-life isused, if the isotope is normally contaminated by a second isotope oflonger half-life.

In some instances, the decayed product may still be active as far as anestrogen receptor is concerned and the labeled, but no longer hot,estradiol derivative may block estrogen receptors from receiving the hotsubstrate, thus interfering with the accuracy of any assay or imagingdata obtained. This problem cannot be overcome by using an excess of theestradiol material since doing so may tend to overload the estrogenreceptors and send hot estradiol to other locations, where it may bedetected, generating erroneous conclusions about the presence ofestrogen receptors.

When non-radioactive isotopes are used in chemical syntheses, problemsof low yield and low specificity are often overcome by utilizing largeamounts of starting materials, and labels, and undergoing sufficientpurifications to allow for the isolation of significant amounts of thedesired products. It is clear that this methodology is generallyunacceptable when radioactive labels are used. First, radioactive labelsare usually too expensive for an inefficient synthesis scheme to becommercially utilizable. Secondly, radioactive isotopes can be dangerousand large concentrations of them should be avoided. Also, unreactedstarting materials and undesired side products may be radioactive,causing problems with contamination during clean-up, isolation and wastematerial disposal. Further, if the isolation of the desired producttakes too long there may be problems with decay of the isotope.

Radioactive isotopes of the halogens are generally considered to be themost important types of labels for use in labeling compounds forbiological assays and imaging. The isotopes generally considered to beof most importance and suitable for labelling estradiol according to thepresent invention are fluorine-18 (F-18), bromine-77 (Br-77), iodine-123(I-123) and iodine-125 (I-125).

Iodine-123 labelled estradiol is foreseen to be especially useful byapplicants for all types of tissue imaging where the tissue has"estrogen" receptors. Iodione12 is a gamma emitter having a half-life ofapproximately 13.3 hours. Iodine-123's energy of gamma decay isrelatively high, approximately 159 kilo-electron-volts (KeV). Thetoxicity of iodine is generally well understood, and I-123 is generallyconsidered to be an almost ideal radioisotope for use in biologicalstudies. In particular, its relatively short half-life makesradioactivity contamination a relatively minor problem, while at thesame time, its relatively high energy of decay makes detectionrelatively easy, even in vivo.

In the past, 17-beta-estradiols labeled with I-123 at the 16-positionhave been unavailable in amounts and purities generally considered to beuseful in assays, and other diagnostic work, either in vitro or in vivo,due to problems in their syntheses. Generally, these problems resultfrom the length of time formerly required to introduce an I-123 labelinto the 16-position, stereospecifically, and the length of timerequired to complete the synthesis and isolate and purify the desiredproduct. In addition, those synthetic methodologies which wereconsidered in the past were often of low yield and often resulted in thewaste of large amounts of I-123 label.

The advantages of the present invention, in preparing labeled compounds,will be most apparent if an examination is first made or previouslyknown methods of introducing halo-substituents into the 16-position of17-beta-estradiol. Such an examination, of the major known methods,follows:

A highly publicized method of preparing16-alpha-halo-substituted-17-beta-estradiol is that published by R. B.Hochberg and will be generally referred to as Hochberg's method orsynthesis. The final step of Hochberg's synthesis is shown below andcomprises Iodo-substituents on the 16-beta-bromo-compound, i.e. aFinkelstein reaction: ##STR3##

In the specification and claims of the present application: a wedgeindicates a substituent projecting above the plane of the drawing; adotted line indicates projection below; and, a curve indicates a mixtureof both. These are conventional methods of indicating stereochemistry.Also, Ac is used to indicate an acetyl group, --C(O)CH₃.

The substitution reaction is generally run in 2-butanone for anywherefrom 12 to 24 hours. Although the reaction has been utilized tointroduce the radioisotope I-125 into the 16-alpha position of17-beta-estradiol, it is generally considered to be of too low a yieldand too long a length of time to allow efficient production of acompound by the substitution of a radioisotope, such as I-123, which hasa relatively short half-life.

Even if conditions are found which allow for an increase in the rate ofthe substitution reaction, it appears unlikely that the synthesis andpurification method proposed by Hochberg can be readily and economicallyutilized to prepare commercially useful 16-alpha-substituted radioactiveestradiol derivatives when the radioactive isotope has a very shorthalf-life. For example, the Hochberg synthesis may requiretime-consuming product isolations and purifications. Further, thereaction does not appear to be adaptable to substantially stereospecificpreparation of 16-beta-substituted compound.

The following scheme shows the overall Hochberg method of synthesis:##STR4##

There are certain problems with the above reaction scheme. For example,the bromination step yields two compounds, mostly the beta form, and thereduction step gives a mixture of all four possible bromohydrins, fromwhich the desired isomer has to be isolated. No single intermediate isformed from which the 16-alpha-substituted compound can be rapidlyformed and from which the 16-beta-substituted compound can also berapidly formed. Also, the starting material for the final Finkelstein,16-beta-Br-17-beta-estradiol, and the product of the final substitution,16-alpha-I-17-beta-estradiol, have similar characteristics forchromatographic purposes, so their clean separation from one another, inthe event that the final substitution does not go to completion, can bedifficult. Further, during the Finkelstein reaction and work-up theremay be epimerization of starting material or product, thus decreasingefficiency. Also, one epimerization product,16alpha-Br-17-beta-estradiol, has very similar chromatographicproperties to the desired product, making purification somewhatdifficult.

Accordingly, researchers' initial attempts to form 16alpha-¹²⁵I-17-beta-estradiol via the Hochberg method resulted in products havespecific activities which were very low, approximately 95 to 140 Curiesper millimole (Ci/mole), as opposed to the theoretical specific activityof approximately 2,000 Ci/mmole. It is reported in the literature thatmeticulous purification of the 16-beta-Br-17-beta-estradiol used in theFinkelstein reaction has resulted in some increased specific activity;however, purity of products still appears to be a problem and seems tokeep specific activity down.

Another, practical, problem is associated with Hochberg's method. Mostradioactive halogen anions are commercially available in the form of anammonium salt or a sodium salt. In the case of radioactive iodides bothammonium salts and sodium salts are usually available whereas, in thecase of the bromides and fluorides, usually only the sodium salt isavailable. These radioisotopes are normally shipped in water, which doesnot readily evaporate, and significant amounts of base, either sodiumhydroxide or ammonium hydroxide, will be present. The Finkelsteinreaction, and its starting material, may be sensitive to the presence ofeither base or water. Therefore, the commercially available isotope, inits basic storage mixture, usually must be scrupulously neutralized anddried before it can be utilized. It is evident that it would bedesirable to develop a reaction methadology which is at least relativelyinsensitive to the presence of water and preferably can tolerate base.

An alternative method of labeling 17-beta-estradiol with halogens at the16-position has been developed and utilized by Katzenellenbogen et al.Their general reaction methodology is shown below: ##STR5##

Katzenellenbogen et al. J. Med. Chem., 23, p. 994-1002 (1980) describebromination of the enol acetate (1) followed by hydrolysis to give16-alpha-Br-3-hydroxy-1,3,5(10)-triene-17-one(16-alpha-Br-estrone; (2b)as proceding with relatively high yield and relatively high specificityfor the alpha-product (2b). Formation of the 16-beta-Br-compound, 4a,was accomplished by epimerization of the 16-alpha-compound (2b) in acid.The epimeric ratio was 1/1.8 (2b/4a). In commercial use, such a mixturewould have to be separated by chromatography and clean separation can beexpected to be difficult and time-consuming.

16-alpha-Br-17-beta-estradiol (3a) was formed from reduction of theketone (2a) with lithium aluminum hydride. (LiAlH₄). The reductionyields both the 17-alpha-and 17-beta-alcohols (3a and 3b) in a ratio of2/1 (17-beta/17-alpha; 3a/3b). Such a mixture can, in theory, beseparated by chromatography; however, again, since the products are sosimilar, chromatographic separation may be expected to be difficult andtime-consuming.

Katzenellenbogen prepares Hochberg's precursor, compound 5 above, byreduction of a mixture of 16-alphaand 16-beta-Br-estrones with zincborohydride (ZnBH₄). All four bromohydrins are formed from such areduction, since the reduction is not stereospecific. Again, althoughchromatographic means may in theory be utilized to separate the fourbromohydrins, they are similar enough in properties so that theseparation can be expected to be very difficult.

16-alpha-C1-estrone (2c) was formed by Katzenellenbogen et al., bytreating the enol acetate (1) with tert-butyl hypochlorite. A mixture ofproducts is generally formed from such a reaction so some purificationis necessary for isolation of the chloride.

16-alpha-C1-17-beta-estradiol (3c) and 16-alpha-C1-17-alpha-estradiol(3d) are formed from treatment of the estrone (2c) with LiAlH₄.Isolation of either product requires purification generally bychromatography, which can be difficult with such similar reactionproducts.

It may be appreciated from the above that the variation of the Hochbergmethod utilized by Katzenellenbogen et al. is generally inefficient for16-substituted compounds, especially when radioisotopes are being used.First, in nearly all instances, product mixtures are formed, whichwastes expensive label and which can be hard to separate. Also, too muchtime may be needed to easily handle radioisotopes of relatively lowhalf-lives.

Longcope et al. have developed a method of synthesis for16-beta-I-17-beta-estradiol which is significantly different fromHochberg's synthesis and the Katzenellenbogen variations. Theirsynthesis begins with 16-alpha-17-beta-estriol and is shown below:##STR6##

The Longcope method yields a product mixture which includes the desiredbeta-product together with an elimination product, so, again, it isinefficient. Secondly, it cannot be readily adapted for formation of16-alpha products. Also, radioactive triphenylphosphite methiodide isexpected to be difficult to prepare. In addition, it does not appear tobe easily adaptable to the utilization of other halogens and theirradioactive isotopes.

The above three general synthetic methodologies illustrate many of theproblems associated with syntheses of 16-substituted, either alpha- orbeta-, 17-betaestradiols. Generally, reaction mixtures includingnumerous products are formed. Also, no synthesis is readily adaptable toyield, stereospecifically, either the 16-alpha or the 16-beta isomer asdesired. Also, the lengths of time required for the introduction ofhalogen label and isolation of the products tend to make the utilizationof the above schemes for radioactive isotopes, especially isotopes withrelatively short half-lives, very difficult, if not commerciallyimpossible.

OBJECTS OF THE INVENTION

The principal objects of the present invention are: to provide a methodof rapidly introducing a radioactive halogen, particularly ¹²³ I, intoestradiol; to provide a general method of preparation of16-halo-substituted-17-beta-estradiols; to provide such a method bywhich radioactive substituents, of relatively short half-lives, can bequickly and efficiently introduced into the 16-position of17-beta-estradiols; to provide such a method by which substitution ofthe substituent into the 16-position of 17-beta-estradiols can be madeeither alpha or beta as desired, either substitution being made withnearly complete stereospecificity; to provide a synthetic intermediatefor use in such a method of synthesis from which either16-alpha-substituted-,or, 16-betasubstituted17-beta-estradiols can berelatively quickly, stereospecifically and efficiently derived; toprovide a method by which 16-alpha-¹²³ I-17-beta-estradiol, having arelatively high specific activity, suitable for use in estrogen receptorassays and imaging studies can be quickly, efficiently, and easilymanufactured; to provide a method of synthesis by which 16-beta-¹²³I-17-beta-estradiol, having a relatively high specific activity andbeing suitable for use in estrogen receptor assays and imaging studiescan be relatively quickly, stereospecifically and efficientlymanufactured; to provide the products of such synthesis; to provide thespecific product 16-alpha-¹²³ I-17-beta-estradiol having a specificactivity of greater than 2000 and preferably at least 5,000 Ci/mmole andbeing collected in sufficient amounts for utilization in estrogenreceptor assays and imaging studies; to provide the specific product16-beta-¹²³ I-17-beta-estradiol; and to provide such methods ofsyntheses which are relatively easy to utilize, are economical and whichare particularly well suited for their proposed usages.

Other objects and advantages of the present invention will becomeapparent from the following descriptions, wherein are set forth by wayof illustration and example certain embodiments of the presentinvention. As required, detailed embodiments and examples of the presentinvention are disclosed herein. However, it is to be understood that thedisclosed embodiments and examples are merely exemplary of theinvention, which may be embodied in various forms. Therefore, specificdetails disclosed herein are not to be interpreted as limiting butrather merely as a basis of the claims and a representative basis forteaching those skilled in the art to variously employ the presentinvention.

SUMMARY OF THE INVENTION

It will be understood that certain superscripts used herein are alsoused in the claims. The superscripts are generally used consistentlybetween the specification and claims. Therefor, the superscripts may notappear in numerical order in the claims or the specification.

The present invention is directed to a new synthetic method forpreparing 16-halogen-substituted-17-beta-estradiols. In particular, asynthetic scheme is presented which permits the introduction ofIodine-123 (I-123) into either the 16-alpha- or 16-beta-position of17-beta-estradiol, essentially stereospecifically, as desired. Thenature of the synthesis is such that the desired product can beisolated, in relatively large and useful amounts, before the I-123label, which has a relatively short half-life of approximately 13.3hours, has decayed to such a point that the product mixture is no longerhot enough to be desirable for use in biological assays. Generally,I-123 can be introduced into 17-beta-estradiol in approximately one totwo hours utilizing methods described herein such that the decayhalf-life of 13.3 hours is readily accommodated and excessive decay doesnot occur before the product can be beneficially utilized.

Also, it is foreseen that the general synthetic scheme developed isreadily adaptable to the syntheses of other16-substituted-17-beta-estradiol products. For example, it isanticipated tha 16-substituted fluorine, chlorine and brominederivatives can be relatively quickly formed. Accordingly, it isforeseen that radioisotopes of the various halogens can be rapidlyintroduced into the 16-position of 17-beta-estradiols.

The synthetic scheme developed is readily adaptable for the highlystereospecific synthesis of either16-alpha-halo-substituted-17-beta-estradiols or16-beta-halo-substituted-17-beta-estradiols. In particular, a productsolution including substantially only16-alpha-halo-substituted-17-beta-estradiol can be obtained. Also, aproduct solution including substantially only16-beta-halo-substituted-17-beta-estradiol can be obtained via a stepmodification in the reaction scheme. The term "stereospecific", as usedherein, means that the product mixture includes substantially only thedesired stereoisomer, be it 16-alpha or 16-beta.

The following reaction scheme outlines a general synthesis of either16-alpha-halo-substituted or 16-beta-halo-substituted-17-beta-estradiolsaccording to the present invention: ##STR7## WHEREIN: Ac is C(O)CH₃

AY is a slat of a halide with:

Y being halide ion; and A being cation

For ease of reference the intermediates and products in the reactionscheme will be referred to, as indicated above, beginning with RomanNumeral X.

An important step in the synthesis involves the utilization of asynthetic intermediate XIV, a 16-beta-triflate-estradiol in which boththe 3- and 17-beta-hydroxy groups have been protected with an acetategroup. This intermediate is relatively stable and can be easily purifiedby high pressure liquid chromatography (HPLC). Also, it is relativelyeasily characterized and is relatively stable to the presence of water.

As shown in the reaction scheme, according to one series of reactions,the triflate diacetate XIV is hydrolized in acid to yield at16-beta-triflate of 17-beta-estradiol, XX. Following this, the diol XXis treated with a nucleophile source, AY, to yield a product XXI inwhich the halogen substituent, Y, is located alpha on the 16-position.This latter substitution reaction occurs relatively rapidly, as will beseen from the examples below, and in generally high enough yield to makethe synthesis commercially utilizable. Also, the substitution, which isbelieved to proceed via an S_(N) 2-type mechanism, is generally observedto be relatively stereospecific.

Also as seen in the above reaction scheme, a16-beta-halo-substituted-17-beta-estradiol derivative XVI can also bereadily prepared from the same triflate intermediate XIV. According tothe method of the invention, if the triflate diacetate XIV is reactedwith a halide anion source AY, before hydrolysis to an estradiol, a16-beta-halo-substituted-17-beta-estradiol, 3,17-beta-diacetate (XV) isformed. While the mechanism for this substitution is not fullyunderstood, the reaction appears to be highly stereospecific and yieldsalmost exclusively the 16-beta-halo-substituted product (XV). Presumablythe 17-beta protecting group interacts with the triflate group at the16-beta position to cause the substitution to occur in the mannerobserved.

If the diacetate product, XV, is then hydrolized,16-beta-halo-substituted-17-beta-estradiol, XVI is readily isolated.

Numerous features of the above mentioned substitutions and hydrolysesmake the synthetic scheme of the present invention highly advantageous.First, the 16-substituted halo-compounds are readily separated from the16-beta-triflates, regardless of whether dihydroxy or diacetal, so theproducts from the substitutions can be easily separated from thestarting material. This is due to the large difference inchromatographic properties between triflates and halogen compounds.

Secondly, the substitution reactions are relatively rapid so anions ofisotopes which have relatively short half-lives can be utilized as thenucleophiles displacing the triflate group. In part, this is probablydue to the nature of the triflate anion as a very good leaving group forsubstitution reactions.

Also, as mentioned above, the triflate group and the halo-substitutionreactions thereon have been found to be relatively stable to thepresence of water in the reaction mixture. It was also noted above thatradioisotopes are normally only commercially available in aqueoussuspension. Thus, the insensitivity of the triflate to water means thatscrupulous drying of the isotope reagent is not necessary. However, somedrying is preferred and normally is conducted.

The substitutions of the triflate group preferably utilize appropriatecrown ether with the nucleophile.

The general source of radioactive halide ions is the sodium halide orthe ammonium halide slat. It has been found that a useful crown eitherfor complexing with the sodium and ammonium cations is 18-Crown-6. Inthe syntheses of the radio-labeled estradiols discussed herein, thecrown ether is normally utilized in excess, since a very small amount ofthe halide is present in the reaction mixture. The excess of crown etherhas not been found to cause any complications in the syntheses.

Another advantage to the present scheme is that for thehalo-substitution of the triflate anion, the conditions are such thatepimerizations of the products are kept to a minimum. Generally, thetriflate anion is a very poor nucleophile and the conditions are suchthat epimerization is unlikely. By "poor nucleophile", it is meant thatrelative to the halogens, the poor nucleophile is unlikely to attacksubstrate and cause multiple substitutions. A result of this is that inaddition to the fact that the mechanisms of the substitution reactionsare highly stereospecific, to yield virtually only the desiredstereoisomer, the reaction conditions and product mixtures are such thatepimerizations, which lead to undesired products, are generally avoided.In Finkelstein reactions, by contrast, epimerizations of startingmaterials and products can be a problem.

It is apparent that the 16-beta-triflate ofestradiol,3,17-beta-diacetate (XIV) is a very useful intermediate forthe introduction of halogens, especially iodine, stereospecifically,into the 16-position of estradiol. It is readily seen that the disclosedsynthesis of this triflate as an intermediate or precursor for thesyntheses of the desired products is very useful and important. It willbe understood that when radioactive substituents of relatively shorthalf-lives are used, the triflate intermediate may be prepared and sentto a location near a facility where the use of the radioactivity labeledestradiol is to take place. The halogen substitution reaction discussedabove would then be performed at this location, the product purified,and the labeled estradiol would then be ready for use, before theradioisotope has had a chance to decay significantly.

It is foreseen that certain pharmaceutical compositions including a16-¹²³ I-17-beta-estradiol may be highly useful in imaging tissue withestrogen receptors located therein. Generally, for imaging compoundswith a short half life and high specific activity, especially a specificactivity greater than 5,000 curies per millimole, are preferred. Forexample, a mixture of ethanol and aqueous saline solution may be used.For imaging in humans, it is foreseen that a preferred injectedconcentration of the radioactive estradiol component in the dilutent isbetween 1.0 and 10.0 millicuries per millimeter, so that all of theradioactive compound may be injected in a relatively small volume.

A general synthesis of a triflate, XIV, was outlined in the reactionscheme illustrated above. The starting material, estrone (X) is readilyavailable and upon treatment with isopropenyl acetate in acid readilyforms an enol acetate XI.

The next step in the synthesis, that is, the oxidation with a peroxyacid to yield ketone XII, is both critical and somewhat unexpected. Upontreatment with meta-chloroperbenzoic acid, under base conditions, i.e.with sodium bicarbonate, enolate XI was observed to form ketone XII.This is in contrast to the chemical literature which suggests thatoxidation of enolate XI with peroxy acids will form relatively stableepoxides. It will be understood that the isolation of ketone XII fromthe reaction with the peroxy acid facilitates a simple synthesis of thedesired triflate XIV, since the oxidation yields only the17-beta-acetate shown.

The next step in the synthesis, the reduction of the ketone XII to formalcohol XIII is performed with sodium borohydride (NaBH₄) and isobserved to be rapid, of relatively high yield and highlystereospecific, giving reduction only from the alpha face to yield the16-beta-hydroxy compound XIII. When lithium aluminum hydride (LiAlH₄) isused as the reducing agent, a mixture of alcohols is formed. It isbelieved that the reduction is controlled by steric factors and as longas reagent which is primarily sensitive to steric approach to reductionis utilized, the amount of alpha attack can be maximized and essentiallyonly the 16-beta-hydroxy compound isolated.

The next step in the synthesis of the triflate XIV is formation thereoffrom the hydroxy compound XIII. This is readily achieved with triflicanhydride, (CF₃ SO₂)₂ in the presence of pyridine. As reported above,the triflate XIV is relatively stable and can be easily purified byHPLC. THus, if any undesired side products are present from the previousreaction steps, they can be easily separated from the desired triflateproduct.

It is foreseen that the above synthesis is a generally useful, syntheticscheme for the generally stereospecific production of certain16-halo-substituted-estradiols.

Beginning with estrone (XXX), the first part of the overall synthesisinvolves conversion to an enolate and protection of the 3-hydroxy group.In general: ##STR8##

In the specific example discussed earlier R^(XI) and R^(XII) were bothacetyl groups. However, it is foreseen that other groups may be utilizedand R^(XI) and R^(XII) need not be identical. For example, the 3-hydroxygroup of estrone XXX might be protected by conversion to an ether orester other than acetate before the 17-keto group is converted to anenolate. What is required, generally, is that R^(XII) be ahydroxy-protecting group which can be readily hydrolyzed with acid forlater removal and which is stable to the conditions of oxidation of the17-enolate group. It is expected that certain silyl ethers, and estersother than acetates, may be used.

It is foreseen that under certain circumstances it may be desirable toleave the 3-hydroxy group unprotected, in which case R^(XII) is H. It isforeseen that the presence of an -OH group at the 3-position will notsignificantly interfere with the later reactions, although it may beconverted to an ester, for example a triflate, when the acidanhydride/pyridine or analogous reaction is run. This, however, is notforeseen to be a problem since whatever ester group is placed on the3-position is likely to be easily removable during the final acidhydrolysis. Also, during the hydride reduction step, if the 3-hydroxygroup is not protected, it may react with the hydride reagent, however,it is foreseen that this should not be a substantial problem if anexcess of hydride reagent is used.

Protecting group R^(XI), on the other hand, is an enolate protectinggroup of epoxide rearranging proclivity. Its general characteristics arethat it can be used to trap an enol, as an enolate, and it can beremoved by treatment with acid, once the enolate has been converted to aprotected hydroxy group as in the next step. Also for the presentpurpose, group R^(XI), should be able to function in the followingoxidation and rearrangement: ##STR9##

The mechanism of the oxidation/rearrangement reaction is not fullyunderstood, however it appears to yield, substantiallystereospecifically, the 17-beat-16-keto compound XXXII. It is foreseenthat enolate protecting groups other than --C(O)CH₃ may be utilized asR^(XI).

The oxidation, [O], described above was conducted with meta-chloroperbenzoic acid (m-CPBA) as the oxidizing reagent. It is foreseen thatother epoxide forming oxidizing reagents may be used, especially otherperoxy acids. The conditions of the m-CPBA oxidations were generallybasic with NaHCO₃, to protect the groups --OR^(XI) and --OR^(XII) fromhydrolysis. Generally, neutral or basic conditions for the oxidation arepreferred.

The next major step in the syntheses of 16-halo-substituted estradiols,according to the present invention, is the reduction, [R], of the16-keto-3,17-beta-diprotected substrate (XXXIII), to the16-beta-hydroxycompound (XXXIV): ##STR10##

In most instances, the groups R^(V) and R^(VI) will be the same asR^(XI) and R^(XII) respectively. However, it is not necessary that theybe so. It is foreseen that compound XXII could be converted to acompound XXXIII, having a different pair of protecting groups, throughdeprotection of the 3,17-beta-hydroxy groups and re-protection withprotecting groups R^(VI) and R^(V). Such a conversion may be desirableto maximize certain aspects of either the preceding or followingreactions, or to aid in the syntheses of derivatives with functionalgroups, not discussed herein, located at other positions in thesubstrate. It is foreseen that R^(VI) may be almost any protecting groupwhich can be readily removed by conventional acid hydrolysis techniques;and, R^(V) is preferably a protecting group which, in addition, will notinterfere with the direction of hydride attack during the reduction.Also, --OR^(V) is preferably a relatively poor leaving group so it willnot be displaced during reduction.

As mentioned above, the reduction [R] is accomplished with a hydridesource or hydride reducing agent. Preferably, a hydride reagent is usedwhich gives exclusive or generally exclusive alpha-attack to yieldsubstantially only the 16-beta-hydroxy compound XXXIV upon aqueousworkup. NaBH₄ has been found to work well, while LiAlH₄ gives a mixture.It is believed that reducing agents which are principally affected bysteric factors will tend to maximize alpha-hydride attack.

The next general step in the reaction sequence is conversion of the16-beta-hydroxy group into an appropriate leaving group for displacementby the halide ions: ##STR11##

It will be understood that R and R^(I), in XXXV, need not be identicalto R^(V) and R^(VI), respectively, in XXXIV, although generally theywill be. R^(I) is preferably a protecting group which is readilyhydrolyzed with acid. R is preferably a readily hydrolyzable protectinggroup which does not interfere with the formation of --OR^(II).Generally this requires --OR to be relatively non-bulky. Also, --OR, ifcompound XXVI is to be used for the halogen-displacement of --OR^(II) atC-16, is preferably a poor enough leaving group so that substitution atC-17 will not compete with substitution at C-16 in the next step. It isbelieved that if --OR is a carboxy group, this latter requirement willgenerally be fulfilled.

On the other hand, --OR^(II) is preferably an excellent leaving group insubstitution reactions. The currently preferred group is a triflategroup, --OSO₂ CF₃, formed from reaction of the 16-alcohol, XXXV, withtriflic anhydride and a base, preferably pyridine. Generally, a weakbase at low nucleophilicity is preferred so that completing substitutionand elimination reactions are minimized. In general, --OR^(II) willusually be an ester leaving group, generally an ester of a sulfonicacid. It is preferred that the group --OR^(II) not be too readilyhydrolyzable or deprotection may occur before desired.

If the 16-alpha-halo compound is desired, the next step in the synthesisis as follows: ##STR12##

If R is a protecting group which is non-beta directing, that is, it willallow nucleophilic attach at C-16 to proceed with inversion, then thesubstitution reaction with AY can be conducted on the protected compoundXXVI. It will be recalled, however, that when --OR is --OC(O)CH₃, attackon XXVI, at least when --OR^(II) is a triflate, proceeds with retentionat C-16. Under these conditions, compound XXXVI should be hydrolyzed todeprotect at C-17 and give XXXVII. It is expected that, if a protectinggroup was left on the 3-hydroxy substituent, the substitution reactionwill proceed with inversion also. However, in most circumstances,hydrolysis is C-17 will be sufficient to deprotect at C-3 as well.

It will be understood that differing combinations of --OR and --OR^(II)will yield attack with inversion. If --OR is --OAc and --OR^(II) istriflate, then attack with AY, where AY is NH₄ I or NaI, proceeds withinversion at C-16.

If the 16-beta-halo-substituted compound is desired, the reaction willbe as follows: ##STR13##

In XL, --OR^(III) is a protected hydroxy with beta-directingcapabilities, when associated with --OR^(II). Such a combination, asstated above, is when --OR^(III) is -OAc and --OR^(II) is --OSO₂ CF₃. Itis foreseen that other combinations with similar chemical features mayalso be used. The group --OR^(III) may be a group --OR^(II) from thepreviously discussed series.

It will be understood that hydrolyses of the protected C-3 and C-17groups may be conducted, whenever appropriate, for the desired diols tobe formed. These will be generally acid hydrolyses so that thehalo-substituent at C-16 will be undisturbed.

The following examples illustrate the high efficiency and versatility ofthe present invention in application to form certain 16-substitutedestradiols. In addition, the following examples are for purposes ofillustration of the invention and should not be interpreted as limitingthe scope of applicants' invention.

EXPERIMENT 1 Synthesis of the 16-beta-triflate of Estradiol,3,17-beta-diacetate (XIV)

A. Formation of the Enol Acetate (XI). ##STR14##

A solution of 10.0 grams (g.) (37.0 millimoles) of estrone (x) wasdissolved in 70 milliliters (ml.) of isopropenyl acetate and 10 ml. ofcatalyst solution. The catalyst solution comprised 0.2 ml. ofconcentrated H₂ SO₄ dissolved in 10 ml. of isopropenyl acetate. Thereaction mixture was heated to boiling and approximately 10 ml ofdistillate was taken off over a period of 0.5 hours. An additional 30ml. of isopropenyl acetate was added along with approximately 1 ml ofcatalyst solution and boiling was continued until approximately 50 ml.of distillate was taken off. At this time another 30 ml. of isopropenylacetate and 1 ml. of catalyst solution was added and boiling continueduntil a second 30 ml. of distillate was taken off.

The reaction mixture was cooled to room temperature and dilluted with200 ml. of ether. The ether solution was washed with cold concentratedsodium bicarbonate solution The solution was then washed with water,dried and the solvent was evaporated. The residue was taken up in 50 ml.of acetone and passed through a silica gel column (500 grams in a 5 cm.by 30 cm. column) developed with hexane acetone 4/1. Upon removal fromthe column, the solid was recrystallized from ethanol (120 ml.). Theproduct was a yellow solid (9,88 grams, 75% yield, melting point 145 to148 degrees centigrade). An NMR spectrum was run of the resultingproduct and it was consistent with the structure for the desired product(XI).

B. Oxidation of the Enolate (XI) to Form 16-keto-estradiol,3,17-beta-diacetate (XII). ##STR15##

A 0.5 millimolar aqueous sodium bicarbonate solution was prepared and100 ml. of the solution was added to 150 ml. of chloroform in a 500 ml.round bottom flask. The diacetate (XII) (9.88 grams, 27.9 millimoles)was added to the chloroform solution in one portion. After the steroidhad dissolved, 6.90 grams (40.0 millimoles) of m-chloroperoxyperbenzoicacid was added in one portion and the reaction mixture was stirred for15 hours at room temperature. The aqueous phase was then removed in aseparatory funnel and the organic phase was washed with two 50 ml.portions of 10% sodium bisulfate solution and then two 50 ml. portionsof saturated aqueous sodium bicarbonate solution. The organic solutionwas dried over sodium sulfate, filtered, and evaporated to dryness togive a yellow semisolid which was recrystallized from enthanol to give awhite solid (5.50 grams, 53% yield, melting point 146 to 149 degreescentigrade.) This solid was recrystallized again from ethanol to give awhite solid (3.54 grams, 34% yield, melting point 151 to 153 degreescentigrade, [alpha]_(D) =54.5 (EtOH), one spot by TLC (hexane: acetone,4/1, silica gel) (NMR analysis confirmed that only isomer XII wasformed.)

C. Reduction of Ketone XII to Form 16-beta-hydroxy-17-beta-estradiol,3,17-beta-diacetate XIII. ##STR16##

The 16-oxo-estradiaol-3,17-diacetate (XII) (1.04 grams, 2.81 millimoles)was dissolved in 80 ml. of isopropanol and 35 milligrams (0.92millimoles) of sodium borohydride was added in one portion at roomtemperature. After 0.5 hours the reaction mixture was treated with 3drops of concentrated hydrochloric acid and evaporated to dryness. Theresidue was purified by preparative TLC (hexane:acetone, 3:1, silicagel, eight 1,000 microplates) with the highest R_(f) component elutedand isolated as a white solid (144 milligrams, 14% yield, melting point75 to 80 degrees centigrade; NMR in CDCl₃, consistent with the abovestated structure XIII).

D. Formation of Triflate XIV from Diacetate XIII. ##STR17##

Fifty-four milligrams (0.145 millimoles) of the16-beta-17-beta-estriol-3,17-diacetate (XIII) was dissolved in 0.5 ml.of deutero-chloroform in a septum top vial and 21 microliters (21milligrams, 0.260 millimoles) of pyridine was added. The vial was cooledin an ice bath and 40 microliters (65 milligrams, 0.237 millimoles) oftriflic anhydride was added via syringe. After one hour the reactionmixture was filtered and the filtrate purified by preparative TLC(hexane:acetone, 3:1 silica gel, one 1,000 microplate) to give acolorless solid (59 milligrams, 81% yield, melting point 50 to 58degrees; NMR in CDCl₃, consistent with the above stated structure XIV).

The deprotected diol is readily isolated from the diacetate byhydrolysis with concentrated HCl in tertbutanol (t-BuOH) analogously tothe hydrolysis conducted in experiment 3 below.

EXPERIMENT 2 Preparation of 16-alpha-¹²³ I-17-beta-estradiol

The radioisotope I-123 is generally available as either NH₄ ¹²³ I orNa¹²³ I. In the experiments described below, NH₄ ¹²³ I was utilized asthe source of the iodine isotope, however analogous experiments havebeen run with Na¹²³ I, which was found to function satisfactorily forthe present purpose. The experimental procedure for either issubstantially identical.

The NH₄ ¹²³ I was received from Atomic Energy of Canada, Ltd (AECL) in asolution of one percent amonium hydroxide. The volume of the solutionwas between 0.5 and 1.0 milliliter (ml). The container was a sealed 10ml. vial with a rubber septum.

The vial was placed in a lead container and equipped with a gas andvacuum line via a 19 guage needle. A 25-guage needle was insertedthrough the septum so that an air flow through the vial could bemaintained. The apparatus was heated, at 80 degrees centigrade, untildryness was obtained in the vial.

An acidic solution of t-butanol was prepared by adding 1 ml. ofconcentrated H₂ SO₄ to 99 ml. of t-butanol. A sufficient amount of theone percent H₂ SO₄ solution was added to the NH₄ ¹²³ I vial toneutralize the sodium hydroxide. This generally required the same volumeof acid solution (0.5 to 1.0 ml.) as the amount of base solution thatwas originally present in the vial. Testing paper was utilized to testthe pH and the acid solution was added until the pH paper showed the pHrange to be between 6.5 and 7.5 or about neutral.

After the above, the amount of radioactivity contained in the solutionwas generally between 8 and 10 millicurries (mCi). Approximatelyone-half of the solution (4 to 5 mCi) was transferred to a 0.30 ml.micro vial fitted with a cap.

200 micrograms of 16-beta-triflate-17-beta-estradiol was dissolved in200 microliters of t-Butanol and transferred to the reaction vialalready containing the NH₄ ¹²³ I. One crystal of 18-crown-6 ether (500to 1,000 micrograms) was added to the reaction solution.

By evaporation with an air stream passing through the vial, the reactionmixture was concentrated to a volume of approximately 100 microliters.At this point, the vial was sealed and heated to boiling for 90 minutes.After heating, the vial was cooled and the contents transferred to amicro-injection vial and injected into an HPLC. The HPLC conditionsutilized are described below.

After the product was collected from the HPLC the solvent was evaporatedby an air stream and the residue dissolved in a 5 percent absoluteethanol/saline solution. The solutions was filtered through a 0.22micron filter for sterilization.

The HPLC conditions were as follows:

1. A Waters model 720 instrument was used in association with a WISP710B automatic injector.

2. The detector was a model 440 UV/Vis absorbence detector with the UVdetector wave length being at 254 or 280 nanometers, as selected.

3. The column was a C18 RP 10 micron, 8 millimeter internal diameter,radial packed column, available from Waters.

4. The solvent system utilized was a 50/50 acetonitrile/water mixturewith the flow rate set at 1.0 ml. per minute.

Under the above conditions, the reaction products came off the columnafter approximately 19 minutes.

When the substitution was conducted, as described above, for between 1and 1.5 hours, the yield of substituted product was about 40-50% (basedon measurements with non-radioactive NH₄ I). When the heating forsubstitution was allowed to continue for an extra 2-3 hours, yields wereincreased to about 70% (again based on experiments with nonradioactivematerials).

Although for some imagery, limits of detection require an estimatedspecific activity of 30,000 Ci/mmole, it is expected that the product,immediately off the column, had a specific activity of near maximum,237,000 Ci/mmole.

EXPERIMENT 3 Preparation of 16-beta-¹²³ I-17-beta-estradiol

Commercially available I-123 in the form of NH₄ ¹²³ I, in one percentsodium hydroxide solution, was neutralized with acid and prepared forreaction with a substrate, in a manner as described above forpreparation of the 16-alpha isomer. The neutralized NH₄ ¹²³ I solutionwas diluted with 500 microliters of t-butanol and approximately one-halfof the solution (4 to 5 milliCuries) was transferred to a micro reactionvial.

Two hundred micrograms of estradiol,16-beta-triflate,3,17-beta-diacetate was dissolved in 200 microliters oft-butanol and transferred to the micro reaction vial. One crystal of18-crown-6 ether (500 to 1,000 micrograms) was added to the reactionsolution. The reaction mixture was concentrated, by drying with an airstream, to a volume of approximately 100 microliters, and was heated toboiling for approximately 90 minutes.

After substitution, 100 microliters of concentrated HCl was added to thesolution and heating was continued for about 60 minutes. After thehydrolysis, the reaction vial was cooled to room temperature and thecontents of the vial were injected to the HPLC system described aboveand the desired product collected from the column.

Yields of the 16-beta-isomer appeared comparable to the 16-alpha-isomerdescribed in Experiment 2 above. The specific activity of the productwas also comparable.

It is to be understood that while certain forms of the present inventionhave been illustrated and described herein, it is not to be limited tothe specific processes, forms, or compositions described.

What is claimed and desired to be secured by Letters Patent is asfollows:
 1. A compound for use as an imaging agent; said compound being16-alpha-¹²³ I-17-beta-estradiol having a specific activity of greaterthan 2,000 Ci/mmole.
 2. The compound according to claim 1 wherein saidspecific activity is at least 5,000 Ci/mmole.
 3. The compositioncomprising the compound according to claim 1 and a pharmaceuticallyacceptable diluent.
 4. A pharmaceutical compound for use as an imagingagent; said compound being 16-¹²³ I-17-beta-estradiol having a specificactivity of greater than 2,000 Ci/mmole.
 5. The compound according toclaim 4 wherein:(a) said estradiol is 16-alpha-¹²³ I-17-beta-estradiol;and (b) said specific activity is at least 5,000 Ci/mmole.
 6. Thecompound according to claim 4 wherein:(a) said estradiol is 16-beta-¹²³I-17-beta-estradiol.
 7. The composition comprising the compoundaccording to claim 4 and a pharmaceutically acceptable diluent.
 8. Thecomposition according to claim 7 wherein said diluent is a mixture ofsaline solution and ethanol.